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Biomedical subjects

Z Shao

Publications and source records attributed to Z Shao.

At least 37 records · Page 2Linked to original sources
PubMed2001

Reconstitution of a functional core polycomb repressive complex.

The opposing actions of polycomb (PcG) and trithorax group (trxG) gene products maintain essential gene expression patterns during Drosophila development. PcG proteins are thought to establish repressive chromatin structures, but the mechanisms by which this occurs are not known. Polycomb repressive complex 1 (PRC1) contains several PcG proteins and inhibits chromatin remodeling by trxG-related SWI/SNF complexes. We have defined a functional core of PRC1 by reconstituting a stable complex using four recombinant PcG proteins. One subunit, PSC, can also inhibit chromatin remodeling on its own. These PcG proteins create a chromatin structure that has normal nucleosome organization and is accessible to nucleases but excludes hSWI/SNF.

Animals↗
PubMed2001

The natural silk spinning process. A nucleation-dependent aggregation mechanism?

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.

Animals↗
PubMed2001

Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution.

The surface structure of actin filaments (F-actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 +/- 1 nm, whereas the smaller inter-filament distance, 8 +/- 1 nm, was consistent with interdigitation of the filaments. The 10 +/- 1 nm F-actin diameter is in good agreement with the results of fibre X-ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F-actin-associated proteins are also present.

Actin Cytoskeleton↗
PubMed2001

Improved precision of quantitating calcium in biological electron probe analysis.

We describe the use of direct multiple least-squares fitting of (unfiltered) biological X-ray spectra. Test of this method on computer-generated spectra and experimental specimens shows that, for the average biological specimen, direct fitting improves the precision of Ca measurements by an approximate factor of 1.6 for a given electron dose. The influence of non-statistical, instrumental sources of measurement errors is considered, and we show a simple method for reducing the effect of these sources of error in the directly fitted spectra.

Animals↗
PubMed2001

Effects of the 20-kilodalton helper protein on Cry1Ac production and spore formation in Bacillus thuringiensis.

Bacillus thuringiensis produces large amounts of various pesticidal proteins during the stationary phase. In order to achieve a high yield and form crystals, some pesticidal proteins require the presence of other proteins. Helper protein P20 is required for efficient production of both the Cyt1A and Cry11A crystal proteins in B. thuringiensis subsp. israelensis. Although full-length Cry1 protoxins are usually independent in terms of expression and crystallization in B. thuringiensis, in this study P20 significantly enhanced production of Cry1Ac protoxin (133 kDa) in an acrystalliferous and plasmid-negative strain. In the presence of P20, the yield of Cry1Ac protoxin increased 2.5-fold, and on average the resulting crystals were 1.85 microm long and 0.85 microm wide, three times the size of the crystals formed in the control lacking P20. Correspondingly, the recombinant strain that coexpressed P20 and Cry1Ac exhibited higher toxicity against Heliothis armigera larvae than the control. Furthermore, serious degradation of Cry1Ac in vivo was observed, which has seldom been reported previously. Actually, most protein was completely degraded during synthesis, and after synthesis about one-third of the expressed protoxins were degraded further before crystallization. In this process, P20 protected only nascent Cry1Ac from degradation, indicating that it acted as a molecular chaperon. In addition, spores were smaller and rounder and had a thinner exosporium layer when they were produced in the presence of P20. In summary, Cry1Ac was severely degraded during synthesis; this degradation was effectively relieved by P20, which resulted in enhanced production. Our results indicated that P20 is an effective tool for optimizing protein production in vivo.

Animals↗
PubMed2001

A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation.

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

Animals↗
PubMed2001

Signal transduction of flumazenil-induced preconditioning in myocytes.

The objective of this study was to examine the role of oxygen radicals, protein kinase C (PKC), and ATP-sensitive K(+) (K(ATP)) channels in mediating flumazenil-produced preconditioning. Chick cardiomyocyte death was quantified using propidium iodide, and oxygen radical generation was assessed using 2',7'-dichlorofluorescin oxidation. Preconditioning was initiated with 10 min of ischemia followed by 10 min of reoxygenation. Alternatively, flumazenil was infused for 10 min and removed 10 min before ischemia. Flumazenil (10 microM) and preconditioning increased oxygen radicals [1,693 +/- 101 (n = 3) and 1,567 +/- 98 (n = 3), respectively, vs. 345 +/- 53 (n = 3) in control] and reduced cell death similarly [22 +/- 3% (n = 5) and 18 +/- 2% (n = 6), respectively, vs. controls 49 +/- 5% (n = 8)]. Protection and increased oxygen radicals by flumazenil were abolished by pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine (800 microM; 52 +/- 10%, n = 6). Specific PKC inhibitors Go-6976 (0.1 microM) and chelerythrine (2 microM), given during ischemia and reoxygenation, blocked flumazenil-produced protection (47 +/- 5%, n = 6). The PKC activator phorbol 12-myristate 13-acetate (0.2 microM), given during ischemia and reoxygenation, reduced cell death similarly to that with flumazenil [17 +/- 4% (n = 6) and 22 +/- 3% (n = 5)]. Finally, 5-hydroxydecanoate (1 mM), a selective mitochondrial K(ATP) channel antagonist given during ischemia and reoxygenation, abolished the protection of flumazenil and phorbol 12-myristate 13-acetate. Thus flumazenil mimics preconditioning to reduce cell death in cardiomyocytes. Oxygen radicals activate mitochondrial K(ATP) channels via PKC during the process.

Animals↗
PubMed2001

Expression and function of c-kit receptor in bone marrow mononuclear cells of patients with myelodysplastic syndromes.

OBJECTIVE: To determine the expression and function of the c-kit receptor in bone marrow mononuclear cells (BMMNC) of patients with myelodysplastic syndromes (MDS). METHODS: Direct immunofluorescence assay and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect c-kit protein and c-kit mRNA expressions in the BMMNC of 29 MDS patients and 10 normal controls. Cell culture was used to detect the function of the c-kit receptor. RESULTS: c-kit protein expression in the MDS group was significantly higher than that in the control group (8.58% +/- 5.28% vs 3.04% +/- 1.49%, P < 0.05). c-kit protein expression in the refractory anemia (RA) group was significantly lower than that in the RA with an excess of blasts (RAEB)/RAEB in transformation (RAEB-t) group (5.12% +/- 2.13% vs 10.01% +/- 5.07%, P < 0.05). The rate of c-kit protein expression was 32.43% in acute myeloblastic leukemia (AML) cases transformed from MDS (t-AML). c-kit mRNA expression in the MDS group was correlated with c-kit protein expression. Interleukin-3 (IL-3) and erythropoietin (Epo), with or without stem cell factor (SCF), upregulated c-kit protein and its mRNA expression. In the presence of IL-3 and Epo, SCF showed significant stimulating effects on the formation of CFU-GM and BFU-E in semi-solid cultures of normal BMMNC, but had no effects on those of the MDS patients. CONCLUSION: The protein and mRNA expression of the c-kit receptor in the BMMNC of MDS patients were higher than those of normal controls, and the function of this receptor in MDS BMMNC was abnormal.

Bone Marrow Cells↗
PubMed2001

[The effectiveness of cyclosporin A in the treatment of autoimmune hemolytic anemia and Evans syndrome].

OBJECTIVE: To test the therapeutic effectiveness of cyclosporin A (CsA) in combination with corticosteroid in the treatment of autoimmune hemolytic anemia(AIHA) and Evans syndrome. METHODS: Forty-four cases of AIHA and Evans syndrome were treated with CsA in combination with a conventional regimen (prednisone + danazol) (18 cases) or conventional regimen alone(26 cases). RESULTS: The complete response rate of CsA group (88.9%) was higher than that of conventional regimen group (57.7%) (P < 0.05), and the relapse rate was lower (3.3% vs 70%) (P < 0.01). CONCLUSION: CsA in combination with conventional regimen can improve the complete response rate and reduce relapse of AIHA and Evans syndrome.

Adolescent↗
PubMed2001

[Immunorelated pancytopenia].

OBJECTIVE: A report of a group of patients with pancytopenia which might be related to abnormal immunity. METHODS: The clinical and laboratory features of 29 patients with positive bone marrow mononuclear cell (BMMNC) Coombs tests were analyzed. RESULTS: Most of these cases were pancytopenia with normal or decreased bone marrow cellularities and increased normoblasts. They were all found to have negative results of conventional hemolysis tests and hematopoietic nutrient determination and had no evidence of malignant clonal hematopoiesis. However, all of them showed positive BMMNC-Coombs test and good response to corticosteroids. CONCLUSION: Immunorelated pancytopenia might be caused by abnormal immunity (especially autoantibody) mediated destruction or abnormal function of blood cells. It is beneficial to differentiate immunorelated pancytopenia from aplastic anemia or myelodysplastic syndromes.

Adolescent↗
PubMed2001

[Sequential intensified immunosuppressive therapy combining with hematopoietic growth factors in the treatment of severe aplastic anemia].

OBJECTIVE: To explore more effective regimen for reducing early mortality of severe aplastic anemia (SAA) and improving therapeutic effectiveness. METHODS: Antilymphocyte globulin/antithymocyte globulin (ALG/ATG) and cyclosporine A (CsA) (sequential intensified immunosuppressive therapy, SIIST), with or without hematopoietic growth factors (HGFs) were administered to 73 SAA patients in a prospective randomized clinical trial to test the effectiveness of the addition of HGFs for the patients. RESULTS: The response rate of SIIST with HGFs group was significantly higher than that of SIIST alone group (89.2% vs 63.9%), with lower rates of early infection (24.3% vs 55.3%) and mortality (4.0% vs 16.7%), shorter duration of cytopenia and blood transfusion dependence and faster recovery of bone marrow hematopoiesis. The addition of HGFs to SIIST was tolerated well in all patients. There was no difference in the treatment outcome of the two groups with GM-CSF plus Epo or G-CSF plus Epo. CONCLUSION: The use of HGFs in combination with SIIST could reduce early infection and mortality rates and, therefore, improve the response rates in SAA patients.

Adolescent↗
PubMed2001

[Study on the transformation from myelodysplastic syndromes into acute leukemias].

OBJECTIVE: To study the patterns of transformation from myelodysplastic syndromes (MDS) into acute leukemias (AL). METHODS: Leukemic transformation of MDS patients was dynamically followed up and the clinical manifestations, peripheral blood and bone marrow pictures, karyotypes, immunophenotypes, response to treatment and prognosis of post MDS acute leukemia (postMDS-AL) were observed. RESULTS: During the past eight year and seven months, 21 (13.91%) of 151 MDS patients progressed to overt leukemia with a median interval of 5 (1 - 21) months. There were no significant differences among the rates of leukemia from RA, RAEB and RAEB-t groups. The transformation was developed either gradually or rapidly. There were five parameters related to the leukemic transformation: under 40 years of age, pancytopenia, more than 0.15 blasts in bone marrow, at least two types of abnormal karyotype and combined chemotherapy. All of the 21 post MDS-AL were acute myeloid leukemia (AML); and most of them were M(2), M(4) and M(5). Two (9.52%) post MDS-AML developed extramedullary infiltration. Leukopenia was found in 47.62% of patients. Two third of the patients, whose bone marrows were generally hypercellular, showed neutropenias. After evolving into AML, 8 (47.06%) patients developed abnormal karyotypes. High expression of immature myeloid antigens, including CD(33) (49.83 +/- 24.50)%, CD(13) (36.38 +/- 33.84)%, monocytic antigen CD(14) (38.50 +/- 24.60)%, and stem cell marker CD(34) (34.67 +/- 30.59)% were found on bone marrow mononuclear cells of post MDS-AML cases. In some cases, lymphoid antigens, such as CD(5), CD(7), CD(9) and CD(19) were coexisted with myeloid antigens. A low complete remission rate (31.25%) and short survival duration with median survival of 6 (1 - 28) months were found in patients with post MDS-AML treated by induction therapy. CONCLUSION: MDS was at high risk of evolving into AML, either gradually or rapidly. Patients with post MDS-AML had specific biologic features and worse prognoses.

Acute Disease↗
PubMed2001

[Establishment and characterization of human inflammatory breast carcinoma neoplasm transplantation in nude mice].

OBJECTIVE: To establish and characterize human inflammatory breast cancer xenograft in nude mice. METHODS: Animal studies, Northern and Western blot, zymograms and immunohistochemistry were used in our studies. RESULTS: Human transplantable inflammatory carcinoma were established in nude mice. This xenograft as its human counterpart exhibited striking erythema of the overlying skin, lympho-vascular invasion and other similar biological characteristics. It also exhibited lung metastasis 4-6 weeks after growth. This xenograft was ER, PR, Her-2/neu negative and p53, EGFR positive. Comparative studies of inflammatory breast cancer xenograft with non-inflammatory xenograft indicated 10-20 fold overexpression of E-cadherin and MUC1. CONCLUSIONS: This human inflammatory breast carcinoma xenograft will provide an excellent animal model to allow us to further dissect out both the upstream regulatory machinery and downstream effector molecules responsible for the inflammatory carcinoma phenotype.

Animals↗
PubMed2000

Single channel properties of neuronal nicotinic ACh receptors in stratum radiatum interneurons of rat hippocampal slices.

The single channel properties of neuronal nicotinic ACh receptors (nAChRs) were investigated in outside-out patches from CA1 stratum radiatum interneurons from thin slices of rat hippocampus. The application of ACh (10 microM to 1 mM) induced the opening of observable single channel currents with two distinct current levels, which corresponded to conductance levels of 38 +/- 3 and 62 +/- 2 pS. The 38 pS channel was observed in 10 % (n = 260) of patches, whereas the 62 pS channel was observed in 4 % of patches; these two channel types were most often observed independently. The alpha7-selective nAChR antagonist methyllycaconitine (MLA; 50 nM) reduced the open probability of the 38 pS channel by 73 %. In contrast, the 62 pS channel was unaffected by MLA, but instead was blocked by dihydro-beta-erythroidine (DHbetaE; 10 microM), a broad spectrum nAChR antagonist. These data suggest that rat hippocampal CA1 stratum radiatum interneurons in the slice possess at least two different types of functional nAChRs, an alpha7-containing subtype with a single channel conductance of 38 pS, and a non-alpha7 subtype with a single channel conductance of 62 pS.

Acetylcholine↗
PubMed2000

A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery.

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.

Bacteriophages↗
PubMed2000

Cryoatomic force microscopy of filamentous actin.

Cryoatomic force microscopy (cryo-AFM) was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filaments are clearly shown to be right-handed helical structures with a periodicity of approximately 38 nm. Even at a moderate concentration ( approximately 10 microg/ml), narrow, branched rafts of actin filaments and larger aggregates have been observed. The resolution achieved is sufficient to resolve actin monomers within the filaments. A closer examination of the images shows that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of approximately 13 nm. The implications of these higher-order structures are discussed in terms of x-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicate that the recently proposed model of left-handed F-actin is likely to be an artifact of preparation and/or low-resolution AFM imaging.

Actin Cytoskeleton↗